Several human tumor nucleolar antigens have been isolated in high states of purity; some exhibit special enzymatic activities. Protein 52/5.3 exhibited "glycogen synthase kinase" activity. Its presence in nucleolar RNP particles suggests that it may phosphorylate specific preribosomal proteins. Another nucleolar phosphoprotein 110/5.2 may, like topoisomerase I, be involved in the uncoiling of nucleolar chromatin. Phosphoprotein 37/5.2 (B23) constitutes up to 5% of the total nucleolar proteins in tumors; its concentration is 6 to 100 times that of normal tissue nucleoli. One peptide that interacts specifically with a monoclonal antibody has been largely sequenced. We are studying the structures and functions of these proteins. Important differences in immunostaining of human tumors and nontumor tissues result from the presence in the growing cells of S-phase-related nucleolar antigens, which are absent from nucleoli of nondividing nongrowing tissues. Antigens PCNA (the proliferating cell nucleolar antigen) and protein 86/6.1 found in nucleoli of tumor cells in G1/S and S-phase are not being purified. Analysis of "difference proteins" of normal and cancer cells from patient material was difficult because of high concentrations of proteolytic enzymes, particularly thiol proteases. We have developed mixtures of multiple inhibitors of proteases and phosphatases that preserve antigens of normal and malignant tissues. Two antigens identified in malignant tumors (100 kilodalton, 68 kilodalton) and not in normal human liver are now being characterized. We now have monoclonal antibodies to several nucleolar proteins including phosphoproteins 110/5.2 (C23), 37/5.2 (B23), and the S-phase nucleolus-localized protein 86/6.1. (AG)